Sequence-specific transcription factors (TFs) regulate gene expression through their interactions with DNA sequences in the genome. These interactions control critical steps in development and responses to environmental stimuli, and their dysfunction can contribute to the progression of various diseases. In this project, as our model system we will examine primary human skeletal muscle myoblasts differentiating into myotubes. In metazoans, regulatory motifs tend to co-occur within stretches of noncoding sequence referred to as cis regulatory modules (CRMs) that regulate expression of the nearby gene(s). In this project, we will assess the physical and functional interactions of CRMs according to their interactions with their immediately adjacent target genes and their effects on reporter gene expression. In particular, we will examine the distant and potentially combinatorial regulatory nature of CRMs, through their physical interactions with the promoters of more distantly located target genes and with each other ('CRM- CRM interactions') and through potentially synergistic effects on regulation of gene expression. The results of this project may reveal trends in how far away are the genes that are regulated by but not adjacent to the regulating CRM. This could have major implications for the prediction and experimental study of the regulatory roles of CRMs, as it is currently unknown how often this phenomenon may occur. The results of this project may also reveal trends in how frequently multiple CRMs work together to regulate their target genes, and what their combined effects are. This could have major implications for the prediction and experimental study of the regulatory roles of CRMs, as currently investigators focus on finding "the [single] CRM" that confers a given expression pattern and assign the result of a reporter assay to the CRM tested on its own, and rarely do not examine it in the context of another candidate CRM with which it may synergize. Finally, the results of this project may reveal whether 'split'CRMs occur (i.e., CRMs composed of physically separated cis regulatory regions that must come together in order to affect gene expression), and may reveal trends in how the different 'parts'of the 'split'CRMs are organized in the genome (i.e., how far apart the 'parts'are, are the parts on the same or different chromosomes, etc.). This could have major implications for the prediction and experimental study of the regulatory roles of CRMs, including our understanding of the evolution of genomic regulatory elements, and would require new computational CRM prediction algorithms to be developed in the future since current algorithms consider CRMs to be contiguous stretches of sequence that function as independent regulatory units. PUBLIC HEALTH RELEVANCE: This project is focused on better understanding the genomic organization of DNA regulatory elements that regulate gene expression. In this project, we will examine differentiating skeletal muscle myoblasts, a biomedically important cell type. The findings from this project will provide a better understanding of gene regulatory mechanisms in these cell types.